The RecBCD enzyme (Exonuclease V) of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. The RecBCD class of enzymes is widely distributed among bacteria (1) but is apparently not present in eukaryotes. The major pathway of double-strand DNA break repair in bacteria involves the coordinated action of nuclease and helicase activities provided by the three-subunit enzyme RecBCD, which is critical for DNA repair(2); recBCD null mutants have reduced cell viability, are hyper-sensitive to DNA damaging agents, and are deficient in homologous recombination involving linear DNA (3-5). Inhibitors of RecBCD would allow us to study the mechanism of this complex helicase-nuclease enzyme. Since this enzyme is crucial for bacterial survival during infection, we anticipate that some of these compounds will, in future work, lead to new, critically needed antibacterial drugs with few off-target effects for human use (6).

Assay Overview:The purpose of this assay is to determine whether compounds identified as active in a set of previous experiments entitled, ""Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of RecBCD (with phage)" (AID 651602), are nonselective inhibitors of the RecBCD due to bacterial cytotoxicity. In this assay, V66 bacteria are not infected with phage but, are incubated in the presence of test compounds alone, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit bacterial growth will reduce well absorbance. Compounds are tested in triplicate at a nominal test concentration of 11.86 uM.Protocol Summary:Prior to the start of the assay, V66 bacterial culture was grown at 37 C until it reached an OD600 of 0.05 or 2.5e07 cfu/mL. V66 was diluted in assay buffer to achieve 1.25e07 cfu/mL. To start the assay, 3 uL of Assay Buffer (0.1% Glycerol + Cation Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 ug/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells.Next, 2 uL of V66 (recBCD+) bacterial culture was dispensed into the appropriate wells. Plates were incubated for 18 hours at 37 C and absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well.The percent inhibition for each compound was calculated as follows:%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )Where:High_Control is defined as wells containing V66 + Ciprofloxacin.Low_Control is defined as wells containing V66 + DMSO.Test_Compound is defined as wells containing V66 in the presence of test compound.PubChem Activity Outcome and Score:The average percent inhibition and standard deviation of each compound tested were calculated. In addition, two values were calculated as well: (1) the average percent inhibition of the wells containing DMSO only, and (2) eight times their standard deviation. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited a percent inhibition greater than the calculated hit cutoff was declared active.The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.List of Reagents:V66 (recBCD+) E. coli bacteria (supplied by Assay Provider)Ciprofloxacin (Sigma, part 17850)Cation-Adjusted Mueller Hinton II Broth (BD, part 297963)1536-well plates (Aurora, part 19326)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was unable to provide all compounds selected for testing in the assay.